Human corneal endothelial cell sheets for transplantation: Thermo-responsive cell culture carriers to meet cell-specific requirements

Accepted Manuscript Human corneal endothelial cell sheets for transplantation: thermo-responsive cell culture carriers to meet cell-specific requirements Juliane Teichmann, Monika Valtink, Stefan Gramm, Mirko Nitschke, Carsten Werner, Richard H.W. Funk, Katrin Engelmann PII: DOI: Reference:

S1742-7061(12)00500-4 http://dx.doi.org/10.1016/j.actbio.2012.10.023 ACTBIO 2443

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

25 June 2012 17 September 2012 17 October 2012

Please cite this article as: Teichmann, J., Valtink, M., Gramm, S., Nitschke, M., Werner, C., Funk, R.H.W., Engelmann, K., Human corneal endothelial cell sheets for transplantation: thermo-responsive cell culture carriers to meet cell-specific requirements, Acta Biomaterialia (2012), doi: http://dx.doi.org/10.1016/j.actbio.2012.10.023

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

revised version - changes are indicated in color

Human corneal endothelial cell sheets for transplantation: thermo-responsive cell culture carriers to meet cell-specific requirements Juliane Teichmanna,b, Monika Valtinka, Stefan Grammb, Mirko Nitschkeb,*, Carsten Wernerb,c, Richard H.W. Funka,c, Katrin Engelmannc,d,* a

Institute of Anatomy, Medical Faculty Carl Gustav Carus, Technische Universität

Dresden, Fetscherstrasse 74, 01307 Dresden, Germany b

Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of

Biomaterials, Hohe Strasse 6, 01069 Dresden, Germany c

CRTD/DFG-Center for Regenerative Therapies Dresden – Cluster of Excellence,

Tatzberg 47/49, 01307 Dresden, Germany d

Department of Ophthalmology, Klinikum Chemnitz gGmbH, Flemmingstrasse 2,

09116 Chemnitz, Germany * corresponding authors: e-mail: [email protected], e-mail: [email protected],

fax: +49 371 33333 223

1

fax: +49 351 4658 533;

Abstract Corneal endothelial diseases lead to severe vision impairment motivating the transplantation of donor corneae or corneal endothelial lamellae, which is however impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. In the current study, we present a novel

thermo-responsive

carrier

based

on

simultaneous

electron

beam

immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces which allows to adjust layer thickness, stiffness, switching amplitude, and functionalization with bioactive molecules to meet cell type specific requirements. We demonstrate the efficacy of this approach for HCECs, which require elaborate cell culture conditions and are strongly adherent to the substratum. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options.

Keywords thermo-responsive polymers, electron beam, cell culture, corneal endothelium, biomolecular functionalization

2

1.

Introduction

Human corneal endothelial cells (HCECs) form a squamous, monolayered epithelium on the posterior side of the cornea, commonly termed the corneal endothelium to distinguish it from the multilayered anterior epithelium of the cornea. They maintain corneal transparency by pumping ions and excess water from the corneal stroma into the anterior chamber [1]. HCECs are arrested in G1-phase of the cell-cycle and show almost no regenerative capacity in vivo. The corneal endothelium experiences a physiological cell loss with age, which is increased by corneal disease, injury or surgical trauma. Cell loss from over 3,000 cells per mm² at birth to below a threshold of about 500 cells per mm² dramatically compromises the overall endothelial pumping capacity [2] and results in vision impairment or blinding of the affected eye due to corneal edema and opacification. Full thickness transplantation (keratoplasty) of long-term cultured (30-37 °C) or short-term cold-stored (4 °C) donor corneas is the gold standard therapeutic option. The endothelial cell density and morphology serves as a sensitive and also the only biomarker to evaluate the quality of a donor cornea. Recently, lamellar endothelial keratoplasty has come into focus and is investigated as an alternative strategy [3]. Lamellae are only comprised of the corneal endothelium, its basal lamina (Descemet’s membrane) and a thin portion of the underlying corneal stroma, and are approx. 100 µm to 200 µm thick [4;5]. Both transplantation techniques require donor tissue with high endothelial cell densities, a demand which is difficult to meet by present organ culture or preservation techniques. A loss of about 20 % of donor tissue due to endothelial cell loss is common in European eye banks [6]. Manual or laser-aided cutting of thin lamellae from donor corneas exerts stretching or thermal forces on the endothelium, so that endothelial cell loss occurring during graft preparation and processing of the tissue 3

remains a major issue. On the other hand, only a thin graft allows for a faster recovery of visual acuity with minimal alteration of corneal refraction [4]. Therefore, visual outcome is inversely dependent on thickness of the graft. Hence, a variety of tissue engineering strategies for corneal endothelium replacement are investigated and many efforts aim at establishing methods for in vitro cultivation and generation of transplantable HCEC sheets [7]. However, HCECs are very sensitive to culturing procedures [8;9] and are also exceedingly adhesive to the cultivation substrate. The use of stimuli responsive polymer (SRP) surfaces [10] may facilitate harvest of HCECs together with their extracellular matrix (ECM) as an intact cell sheet of extreme thinness, an important characteristic with respect to visual outcome after endothelial transplantation in patients. SRPs with a thermally stimulated volume phase transition, like poly(N-isopropylacrylamide) (PNiPAAm) [11] and NiPAAm containing copolymers [12], can be prepared on solid surfaces by different techniques [13]. When immersed in aqueous media, the abrupt change in solubility at the phase transition temperature leads to a reversible swelling and collapsing of the immobilized SRP. This effect was utilized to design switchable cell culture carriers that allow enzyme free harvest of adherent cells or entire cell sheets in general [14] or HCEC sheets in particular [15;16]. However, SRP based cell culture carriers require a balance of cell adhesion for achieving optimal cell growth and effective cell detachment upon stimulation. Compared to the previous work of Sumide et al. [15] and Lai et al. [16] the current study aims at a better understanding of this balance and the respective material parameters where HCECs are employed as a highly relevant example. Cell adhesion, a prerequisite for proper cell and tissue functionality [17], is secured for HCECs by an ECM consisting of laminin, collagen type IV and type VIII and 4

proteoglycans [18]. While adhesion of cells can be facilitated by biofunctionalizing the culture substrate with proteins, detachment of confluent cell sheets often remains a challenge, especially for anchorage-dependent cells like HCECs, which can be strongly adherent [19;20]. We therefore investigated how the properties of a thermoresponsive carrier, that determine cell adhesion and stimulated detachment, can be tuned to meet the requirements of highly fastidious cells such as HCECs. Previously explored approaches to modulate the characteristics of switchable cell culture carriers include the variation of the layer thickness [21], stiffness, degree of swelling and absolute switching amplitude [22], terminal functionalization of SRP chains [23] or inclusion of poly(ethylene glycol) units [24]. Furthermore, immobilization of proteins and peptides [25] was shown to promote initial cell adhesion and proliferation. In the current study, a novel set of cell culture carriers based on poly(vinyl methyl ether) (PVME, exhibiting a distinct phase transition temperature close to cell cultivation temperature [26]) was prepared by simultaneous electron beam crosslinking and grafting of solid polymer films onto polystyrene surfaces followed by biomolecular functionalization [27]. The resulting material platform is demonstrated to be highly tunable and therefore suitable to support cultivation and effective enzyme free harvest of HCEC sheets.

5

2

Materials and Methods

2.1

Preparation and biomolecular functionalization of cell culture carriers

To improve the applicability of analytical techniques, SRP layers were prepared on polystyrene thin films on different supports instead of standard cell culture dishes. Polystyrene thin films (PS, type 148H, BASF, Ludwigshafen, Germany) of about 30 nm thickness were applied by spin coating (solution 1 % wt/wt in toluene, 2000 rpm, 1500 rpm/s, 30 s) onto microscopy cover slips (20 mm in diameter, Menzel Gläser, Braunschweig, Germany) or silicon wafers (15x20 mm2). PS surfaces were treated with air plasma to obtain an appropriate wetting behavior (Harrick Plasma Cleaner PDC-002, 1 min). Subsequently, blends of poly(vinyl methyl ether) (PVME, TCI Europe, Zwijndrecht, Belgium) and the alternating copolymer of vinyl methyl ether and maleic acid (PVMEMA, Sigma-Aldrich, Munich, Germany) were prepared by spin coating (solution 2 % wt/wt or 10 % wt/wt in methanol, 2000 rpm, 1500 rpm/s, 30 s) on the PS surface (referred to as samples with thin or thick SRP layers). A copolymer content of 1% wt/wt or 10% wt/wt led to samples with a different functional group density (referred to as samples with low or high content of binding sites for immobilization of proteins/ peptides). Electron beam irradiation with 150 keV, corresponding to a penetration depth in polymeric materials of about 200 µm, was carried out using the low energy electron facility ADU (Advanced Electron Beams, Wilmington, USA) under nitrogen atmosphere at room temperature (RT). The samples were irradiated with an absorbed dose of 258 kGy or 774 kGy (referred to as samples with low or high degree of cross-linking). The dose was applied stepwise in order to reduce the temperature increase during electron beam treatment. Finally, samples were rinsed in deionized water and ethanol to remove unbound material. 6

Dry thickness of PVME-blend-PVMEMA layers was determined by ellipsometry (SE400adv, Sentech Instruments, Berlin, Germany) on coated silicon wafers. Swelling behavior was characterized by spectroscopic ellipsometry (M-2000VI, J.A. Woollam Co., Inc., Lincoln, USA) as reported in a previous study [27]. Briefly, coated silicon wafers were placed in a liquid media cell (angle of incidence 68°) filled with deionized water (pH 6.5). A computer controlled heating device was used for temperature variation with a rate of 1 K min-1. To calculate thickness and optical properties of the swollen SRP films, fit procedures based on Cauchy multilayer models and an effective medium approximation were applied to the ellipsometric data. The swelling degree was calculated as Q = dT/ddry, were dT corresponds to the swollen film thickness at a given temperature and ddry to the dry film thickness. Mechanical properties of PVME-blend-PVMEMA layers on microscopy cover slips were determined by nanoindentation experiments using an atomic force microscope (AFM, NanoWizard II, JPK Instruments, Berlin, Germany) mounted on an inverted optical microscope (Observer D.1, Zeiss, Jena, Germany). Samples were placed in petri dishes (diameter 35 mm). Measurements were performed in phosphate buffered saline without Mg2+/Ca2+ (PBS w/o Mg2+/Ca2+, Biochrom AG, Berlin, Germany) at 25 °C and 37 °C controlled by a PetriDishHeater (JPK Instruments). A v-shaped cantilever with a nominal spring constant of 0.07 N/m (MLCT, Bruker AFM Probes, Camarillo, USA) was used. Cantilevers were calibrated using the equipartition theorem [28]. Force-distance curves were acquired up to 1 nN contact force and 2 µm/s approach/retract velocity. The Young’s Modulus was extracted from approach force-distance curves using the Herz model (corrected for the use of a quadratic pyramid as intender) provided by AFM data processing software (JPK Instruments).

7

For cell culture experiments samples were sterilized by incubation in 0.02% v/v ProClin®300 Preservative for Diagnostic Reagents (Sigma-Aldrich) in PBS w/o Mg2+/Ca2+ over night at RT. ProClin®300 residuals were removed by rinsing in PBS w/o Mg2+/Ca2+ over night at RT. To allow for covalent protein/ peptide immobilization, maleic acid groups were converted into anhydride moieties in a thermal annealing step (90 °C, over night) [29]. Subsequently, samples were cooled to RT and immediately incubated in a solution of either 10 µg/ml laminin (LN, Sigma-Aldrich) and

10 mg/ml

(Biochrom AG)

chondroitin-6-sulfate or

50 µg/ml

(CS,

Sigma-Aldrich)

in

Medium 199

cyclo(arginine-glycine-asparic acid-D-tyrosine-lysine)

(cRGD, Peptides International, Louisville, USA) in PBS w/o Mg2+/Ca2+ for two hours at 37 °C under sterile conditions. Figure 1 summarizes preparation, biochemical functionalization and structural constitution of the novel cell culture carrier. A hydrophilic polystyrene surface (air plasma treated PS) similar to commercial tissue culture polystyrene (TCP) and a thermo-responsive cell culture system based on the statistical copolymer of N-isopropylacrylamide and diethyleneglycol methacrylate (poly(NiPAAm-co-DEGMA)) [30] developed in a previous study [31] were used as controls. Briefly, a thin film of poly(NiPAAm-co-DEGMA) was prepared on a polymeric surface and subsequently immobilized by low pressure argon plasma treatment [32]. Controls were sterilized and incubated in LN/CS or cRGD as described above to bind proteins or peptides in a physisorptive manner to the surfaces.

8

2.2

Cell culture and analyses

2.2.1 Cultivation and subcultivation of HCECs An immortalized HCEC population (HCEC-12 [33]) was routinely grown in medium F99 (Ham’s F12 Nutrient Mixture/ Medium 199; Biochrom AG), supplemented with 5% v/v fetal calf serum (Biochrom AG), 20 µg/ml ascorbic acid (Sigma-Aldrich), 20 µg/ml recombinant human insulin (Sigma-Aldrich), 10 ng/ml recombinant human basic

fibroblast

growth

factor

(Sigma-Aldrich),

and

antibiotics

(2.5 µg/ml

amphotericin B and 50 µg/ml gentamycin; Biochrom AG). The cells were subcultured at subconfluence using trypsin/ ethylenediaminetetraacetic acid (0.05%/ 0.02% v/v, incubation for 3 min at 37 °C; Sigma-Aldrich), collected in growth medium, centrifuged at 100×g for 5 min and plated at a density of 5× 103 cells per cm2 on culture flasks (growth area 75 cm2, Corning Incorporated, New York, USA) coated with a mixture of 30 µg LN and 30 mg CS in 3 ml PBS w/o Mg2+/Ca2+ (incubation: 30 min, 37 °C). HCECs were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Growth medium was changed three times per week.

2.2.2 Cell adhesion and detachment To maintain the collapsed state of the SRP layers during sample handling and microscopy, the following procedures were performed with pre-warmed (37 °C) PBS w/o Mg2+/Ca2+ or cell culture medium on a customized heating plate. Samples were prepared as described above, rinsed with PBS w/o Mg2+/Ca2+ and incubated in growth medium for 30 min. Thereafter, 5× 104 cells per cm2 were seeded onto the surfaces. HCECs were grown at 37 °C in a humidified atmosphere containing 5% CO2. After four days (average time for monolayer formation on TCP at this seeding density) detachment of the cells was achieved by cooling the samples for 60 min at 9

4 °C.

Adhesion,

proliferation,

monolayer

formation

and

detachment

were

documented by light microscopy (Olympus IX 50 by Olympus GmbH, Hamburg, Germany) with Hoffman Modulation Contrast objective HMC 10 LWDLCA 0.25 na, digital camera AxioCam HR and image analysis software AxioVision 4.7 (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

2.2.3 Cell metabolism Metabolic activity rates of HCECs after four days of growth on SRP surfaces were examined using the cell proliferation reagent WST-1 according to the manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). Supernatants were collected after 30 min incubation with WST-1 supplemented medium at 37 °C in a humidified atmosphere containing 5% CO2. Absorbance was determined at 450 nm (Tecan GENios Microplate Reader and universal data processing software Magellan 6 by Tecan Group Ltd, Männedorf, Switzerland). Absolute cell numbers were determined with a cell counter (CASY-Technology Model TT, Roche Diagnostics GmbH, Innovatis) and related to formazan production. Three replicates were performed and data expressed as mean ± standard deviation.

10

2.2.4 Immunofluorescence microscopy HCECs were cultured on biofunctionalized SRP surfaces for four days as described above. Preparation of the samples for immunofluorescence microscopy was performed under conditions preventing the cells from detaching during staining. Samples were rinsed in 37 °C warm PBS with 100 mg/l MgCl2 6H2O and 100 mg/l CaCl2 (PBS w/ Mg2+/Ca2+, Biochrom AG) and fixed in 37 °C warm 4% wt/v paraformaldehyde (Sigma-Aldrich) in PBS w/ Mg2+/Ca2+ for 15 min at 37 °C. Samples were permeabilized with 0.5% wt/v Triton X-100 (Sigma-Aldrich) in PBS w/ Mg2+/Ca2+ for 10 min at RT. Cell nuclei were stained with 2 µg/ml Hoechst 33342 (trihydrochloride/ trihydrate; Invitrogen Life Technologies, Darmstadt, Germany) in PBS w/ Mg2+/Ca2+ for 10 min at RT. Samples were then blocked by three consecutive incubations in 10% wt/v goat serum albumin (GSA, 60 mg/ml; Dianova GmbH, Hamburg, Germany) in PBS w/ Mg2+/Ca2+ for 10 min at RT, followed by 60 min incubation at RT with one of the following antibodies: a monoclonal mouse antihuman paxillin antibody (1:100; clone 349/ Paxillin; BD Transduction Laboratories, Heidelberg, Germany), a monoclonal mouse anti-human focal adhesion kinase antibody (1:100; clone 77/FAK; BD Transduction Laboratories), a monoclonal mouse anti-human vinculin antibody (1:200; clone hVIN-1; Sigma-Aldrich), a monoclonal mouse anti-human ZO-1 antibody (1:100; clone 1/ZO-1; BD Transduction Laboratories) or a polyclonal rabbit anti-human fibronectin antibody (1:200; Rockland Immunochemicals) in a 1% wt/v bovine serum albumin (BSA, Sigma-Aldrich) solution in PBS w/ Mg2+/Ca2+. Samples were rinsed three times for 10 min with 1% wt/v GSA in PBS w/ Mg2+/Ca2+ and incubated with Alexa Fluor®488 goat anti-mouse IgG or Alexa Fluor®488 goat anti-rabbit IgG (1:200, Invitrogen), and Alexa Fluor®633 phalloidin (1:50, Invitrogen) in a 1% wt/v BSA solution in PBS w/ Mg2+/Ca2+ for 45 min 11

at RT in the dark in order to visualize antibody binding and F-actin filaments of the cellular cytoskeleton. Samples were rinsed with PBS w/ Mg2+/Ca2+, mounted on object slides with anti-fading mounting medium (O. Kindler GmbH, Freiburg, Germany), and viewed by confocal laser scanning microscopy with a Leica TCS SP5, objective HCX PL APO Lbd. Bl 63x/ 1.40-0.60 oil, UV-diode (405 nm), argon laser (488 nm) and helium-neon laser (633 nm) (Leica Microsystems GmbH, Wetzlar, Germany). Image processing was performed with Leica LAS AF software.

12

3

Results

3.1

Preparation of cell culture carriers

Spin coating of PVME-blend-PVMEMA from 2 % wt/wt solution resulted in a dry thickness of ∼ 60 nm (thin SRP layers). Spin coating from 10 % wt/wt solution resulted in a dry thickness of ∼ 600 nm (thick SRP layers). After electron beam irradiation and rinsing in deionized water only 40 to 50 nm and 350 to 400 nm, respectively, were immobilized for samples with an absorbed dose of 258 kGy (low degree of cross-linking). In contrast, all pre-deposited material was immobilized for samples with an absorbed dose of 774 kGy (high degree of cross-linking). The degree of cross-linking determines the temperature dependent swelling behavior. The degree of swelling at 25 °C was found as Q = 3 to 4 or Q = 1.5 to 2 for samples with low or high degree of cross-linking. No swelling occurred at 37 °C (Q = 1) in any of the samples, i.e. water was fully expelled well above the phase transition temperature. Mechanical properties were evaluated by AFM for thick PVME-blendPVMEMA layers (Figure S1). Irrespective of the degree of cross-linking and blend composition, the collapsed layers had a Young’s modulus of 1100 ± 300 kPa at 37 °C. However, at 25 °C layers with a low degree of cross-linking were much softer (180 ± 90 kPa) after swelling than those with a high degree of cross-linking (610 ± 150 kPa).

3.2

Cell adhesion and monolayer formation

As shown in Figure 2, superior adhesion and proliferation was observed for samples with a high degree of cross-linking. Here, formation of confluent monolayers was seen to be independent of SRP thickness and content of binding sites for immobilization of proteins/ peptides (Figure 2 E – H, M – P), although an increasing 13

content of covalently bound protein/ peptide further supported HCEC adhesion and confluent monolayer formation. Particularly, functionalization with cRGD resulted in faster monolayer formation than LN/CS functionalization, where some gaps were observed in the cell layer after the same cultivation time. Contrary, on samples with a low degree of cross-linking, HCEC growth was dependent of SRP thickness and/ or the content of binding sites for immobilization of proteins/

peptides,

again

with

better

results

for

cRGD

than

for

LN/CS

functionalization. HCECs showed moderate to good adhesion and formation of subconfluent to confluent monolayers when SRP layers were thin and had a low content of binding sites for protein/ peptide immobilization independently of the kind of biomolecule (Figure 2 A, B). On samples with a thick SRP layer, and low content of binding sites for protein/ peptide immobilization, monolayer formation was moderate, with cRGD supporting monolayer formation better than LN/CS (Figure 2 C, D). Irrespective of SRP thickness, HCEC adhesion and monolayer formation was also only fair on samples with a high content of binding sites for immobilization of LN/CS, allowing the cells to establish only subconfluent monolayers (Figure 2 I, K), but were improved in case of cRGD (Figure 2 J, L). HCEC adhesion and monolayer formation were very good on the control surfaces poly(NiPAAm-co-DEGMA) and TCP. The cells established a confluent monolayer within four days. Differences in adhesion and monolayer formation regarding the physisorptive protein/ peptide functionalization were hardly discernible (Figure S2).

3.3

Cell detachment

HCEC detachment was enhanced on samples with a low degree of cross-linking (Figure 3). A low content of protein/peptide binding sites and biomolecular 14

functionalization with LN/CS further positively influenced detachment behavior of HCECs (Figure 3 A, B). While detachment was good from thin SRP samples, detachment occurred only partially from samples with a thick SRP layer, leaving the HCEC monolayer perforated and rippled. This effect was stronger on LN/CS functionalized samples, while monolayers cultured on cRGD functionalized samples showed only moderate rippling, but no detachment (Figure 3 C, D). On samples with a low degree of cross-linking and a high content of binding sites for immobilization of proteins/ peptides, monolayers detached only at the edges with marginal folds and opening gaps within the cell layers irrespective of SRP thickness and kind of biomolecule (Figure 3 I – L). Samples with a high degree of cross-linking and a low content of binding sites for immobilization of proteins/ peptides did not support detachment of HCEC sheets irrespective of SRP thickness (Figure 3 E – H). Here, only few cells detached, leaving the adherent monolayer perforated and rippled. Again, this effect was slightly better on LN/CS functionalized samples than on cRGD functionalized samples. Irrespective of protein/ peptide functionalization, HCEC grown on thin SRP layers with a high degree of cross-linking and a high content of binding sites for biofunctionalization did not detach at all (Figure 3 M, N), and formed only folds at the edges when cultured on the respective samples with a thick SRP layer (Figure 3 O, P). Poly(NiPAAm-co-DEGMA) samples with physisorptive LN/CS functionalization allowed for partial detachment of HCEC sheets, while on cRGD functionalized poly(NiPAAm-co-DEGMA) only folding of cell layers at their edges could be observed. No detachment was observed for cells grown on TCP (Figure S3).

15

3.4

Metabolism and morphology

Metabolism. WST-1 assay was performed to examine whether the newly developed surfaces may have a potential adverse influence on HCEC metabolism. It was observed, that metabolic activity rates were comparable on all PVME-blendPVMEMA-based SRP carriers, poly(NiPAAm-co-DEGMA) and TCP (Figure 4). These results corroborate light microscopy observations of cell morphology and cell density. Thus, any adverse impact exerted by the novel SRP system on HCEC metabolism could be excluded. Furthermore, there was a tendency that cRGD functionalized surfaces seemed to support metabolic activity better in comparison to LN/CSfunctionalized surfaces. Morphology. In preliminary tests, it was found that paxillin-containing focal adhesions (FAs) were more often observable in HCECs grown on cRGD coated TCP than on LN/CS coated TCP, but they were generally less detectable with time. With respect to these preliminary results, investigations focused on paxillin staining to gain more information about HCEC adhesion in dependence on the particular surface. HCECs grown on cRGD functionalized surfaces (Figure 5 B, D, F) appeared to develop more spear-head-shaped, paxillin-associated focal adhesions than those grown on LN/CS functionalized surfaces (Figure 5 A, C, E), where paxillin signals were more dotshaped. Prominent paxillin signals were predominantly detected on TCP and poly(NiPAAm-co-DEGMA) (Figure 5 C, D and E, F), while only small and dot-shaped paxillin signals were seen on PVME-blend-PVMEMA surfaces (Figure 5 A, B). Additional staining for fibronectin as an HCEC ECM component revealed, that HCECs cultured on LN/CS functionalized surfaces (Figure 5 G, I, K) developed fibronectin fibrils that were mainly colocalized with the majority of F-actin filaments at the cell edges. Contrasting, cells on cRGD functionalized surfaces (Figure 5 H, J, L) 16

showed more randomly distributed and dot-shaped fibronectin signals. In general, fibronectin signals were more pronounced on TCP and poly(NiPAAm-co-DEGMA) (Figure 5 I, J and K, L) than on PVME-blend-PVMEMA surfaces (Figure 5 G, H). ZO-1 as a typical protein of HCEC tight junctions was detectable in all samples at the lateral cell membranes in association with intercellular contacts (Figure 5 M-R).

17

4

Discussion

The thermo-responsive cell culture carriers introduced in this study allow an independent tuning of physico-chemical and biomolecular characteristics to balance the specific requirements of HCECs for initial cell adhesion and effective detachment of the grown cell layer (Figure 6). The density of anhydride groups used for binding biomolecules was tuned within the SRP layers by taking advantage of the fact that a PVMEMA content of a few percent in the blend does not impair its thermo-responsive properties [27]. The degree of cross-linking, a function of the electron beam parameters, was used to tune swelling behavior and stiffness of the SRP layer as another effective option to adjust the balance between initial cell adhesion and stimulated detachment. Contrary to the well-established electron beam grafting of thin PNiPAAm films from monomer solutions [34], the polymer based approach applied here allows to adjust film properties over a wide range (thickness up to 1 μm with a degree of swelling from 2 to 8) [27]. HCECs sustained a metabolic activity on the PVME-blend-PVMEMA samples comparable with that of HCECs grown on the control samples poly(NiPAAm-coDEGMA) and TCP. Furthermore, ZO-1 as an essential part of tight junctions, a prerequisite for proper HCEC functionality, was detectable in all analyzed samples. It can therefore be concluded, that PVME-blend-PVMEMA can support in vitro cultivation of HCECs while maintaining morphological prerequisites for tissue function. On rigid surfaces cells were previously shown to form FAs in dependence on local stiffness and local internal tension exerted by the cytoskeleton [35-37]. Accordingly, HCECs sense their substratum and respond to its properties by alterations in morphology and migratory behavior [38]. SRP substrate stiffness was modulated by 18

the cross-linking degree and turned out to be the major influencing factor for HCEC attachment. However, while HCECs formed high numbers of paxillin-associated, spear-head shaped FAs on TCP, the cells formed almost no paxillin-associated FAs on PVME-blend-PVMEMA, although both substrates are rather stiff (E ≥ 1 MPa) at 37 °C, pointing at the importance of dissimilar physico-chemical features between the PVME-blend-PVMEMA substrate and TCP for cell-matrix adhesion. On most SRPs, cell detachment is determined by the layer thickness. In the here introduced system, irrespective of layer thickness, cell sheets detached more easily from carriers with low stiffness. Thus, layer stiffness can be used to adjust adhesion and detachment. ECM ligand density was reported to correlate with initial cell adhesion, and promotes cell spreading and the size of FAs in comparison to surfaces without ECM ligands [39;40]. In line with these findings, improved adhesion and monolayer formation were observed for HCECs seeded on substrates with an increasing content of protein/ peptide binding sites, while the ease of cell detachment was gradually impaired. Hence, ligand density is another parameter that can be adjusted to control the balance between cell adhesion and detachment independent of layer stiffness. The natural basement membrane of HCECs is mainly composed of collagen type IV, laminin, fibronectin, and vitronectin [18]. In vitro cultures of HCECs grew better when dishes were coated with LN-5, LN/CS or fibronectin compared to uncoated or gelatine-coated carriers [9;41-43]. Our novel SRP layers were biofunctionalized with LN/CS as natural constituents of HCEC ECM and cRGD as an artificial adhesion ligand. cRGD promoted development of more paxillin-associated, spear-head shaped FAs and actin filaments, while LN/CS supported deposition of fibronectin fibrils in comparison to cRGD. Consequently, cell adhesion was better for cRGD, but detachment of monolayers was inferior to substrates functionalized with LN/CS. 19

Nevertheless, the synthetic adhesion ligand cRGD is preferable for producing transferable HCEC sheets with respect to a future clinical application, because it circumvents the necessity of using natural ECM proteins. The use of other artificial peptides, such as the LN-derived YIGSR, or combinations of peptides may further allow for optimizing the presented SRP-based culture system with respect to cell attachment and detachment of the grown cell sheets [44].

20

5

Conclusions

New techniques in corneal keratoplasty and strategies in tissue engineering require similarly advanced methods for cultivation and harvest of cell sheets for transplantation.

The

here

presented

thermo-responsive

cell

culture

carrier

demonstrates a major step towards this goal: Simultaneous electron beam crosslinking and grafting of PVME-blend-PVMEMA layers with different absorbed doses was applied to adjust material properties including the initial film thickness, degree of swelling and stiffness. Biofunctionalization of the layered carriers with typical ECM constituents or ECM-derived adhesive peptide sequences further improved the flexibility of the approach. The combination of tunable physico-chemical and biofunctional properties was shown to provide a versatile platform for HCEC sheet generation. Non-enzymatic harvest of intact cell layers including their deposited ECM [31] can be expected to aid adhesion of the HCEC graft to the target tissue. In sum, the reported results demonstrate the potential of the introduced approach and motivate the preparation of similar sheets of primary cells required for improved clinical transplantation strategies. Towards this goal future work aims to establish more sophisticated cell sheet manipulation techniques.

21

Acknowledgements This work was supported by the DFG grant EN168/13-1 and the European Regional Development Fund, project 4212/09 15. The authors thank Roland Schulze for ellipsometry measurement, Michael Spaethe for operating the electron beam irradiation setup and Dr. Jens Friedrichs for the AFM experiments. Furthermore the authors thank Dagmar Pette and Tina Kapell for excellent support with the cell culture experiments and Karolina Chwalek for critical reading and discussing the manuscript.

22

References [1] Edelhauser HF. The balance between corneal transparency and edema - The proctor lecture. Invest Ophthalmol Vis Sci 2006;47:1755-1767. [2] Joyce NC. Proliferative capacity of the corneal endothelium. Progress in Retinal and Eye Research 2003;22:359-389. [3] Price MO, Price FW. Endothelial keratoplasty - a review. Clinical and Experimental Ophthalmology 2010;38:128-140. [4] Dapena I, Ham L, Melles GRJ. Endothelial keratoplasty: DSEK/DSAEK or DMEK - the thinner the better? Current Opinion in Ophthalmology 2009;20:299307. [5] Mau K. What DSAEK is going on? An alternative to penetrating keratoplasty for endothelial dysfunction. Optometry-Journal of the American Optometric Association 2009;80:513-523. [6] Armitage WJ, Easty DL. Factors influencing the suitability of organ-cultured corneas for transplantation. Investigative Ophthalmology & Visual Science 1997;38:16-24. [7] Proulx S, Brunette I. Methods being developed for preparation, delivery and transplantation of a tissue-engineered corneal endothelium. Experimental Eye Research 2012;95:68-75. [8] Engelmann K, Bohnke M, Friedl P. Isolation and long-term cultivation of human corneal endothelial cells. Investigative Ophthalmology & Visual Science 1988;29:1656-1662. [9] Engelmann K, Friedl P. Optimization of culture conditions for human corneal endothelial cells. In Vitro Cellular & Developmental Biology 1989;25:1065-1072. [10] Stuart MAC, Huck WTS, Genzer J, Müller M, Ober C, Stamm M et al. Emerging applications

of

stimuli-responsive

polymer

2010;9:101-113. 23

materials.

Nature

Materials

[11] Schild HG. Poly (N-Isopropylacrylamide) - Experiment, Theory and Application. Progress in Polymer Science 1992;17:163-249. [12] Rzaev

ZMO,

Dincer

S,

Piskin

E.

Functional

copolymers

of

N-

isopropylacrylamide for bioengineering applications. Progress in Polymer Science 2007;32:534-595. [13] Da Silva RMP, Mano JF, Reis RL. Smart thermoresponsive coatings and surfaces for tissue engineering: switching cell-material boundaries. Trends in Biotechnology 2007;25:577-583. [14] Elloumi-Hannachi I, Yamato M, Okano T. Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine. Journal of Internal Medicine 2010;267:54-70. [15] Sumide T, Nishida K, Yamato M, Ide T, Hayashida Y, Watanabe K et al. Functional human corneal endothelial cell sheets harvested from temperatureresponsive culture surfaces. Faseb Journal 2006;20:392-394. [16] Lai JY, Chen KH, Hsu WM, Hsiue GH, Lee YH. Bioengineered human corneal endothelium for transplantation. Archives of Ophthalmology 2006;124:14411448. [17] Giancotti FG, Ruoslahti E. Transduction - Integrin signaling. Science 1999;285:1028-1032. [18] Kabosova A, Azar DT, Bannikov GA, Campbell KP, Durbeej M, Ghohestani RF et al. Compositional differences between infant and adult human corneal basement membranes. Invest Ophthalmol Vis Sci 2007;48:4989-4999. [19] Götze T, Valtink M, Nitschke M, Gramm S, Engelmann K, Hanke T et al. Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers. Graefe's Archive for Clinical and Experimental Ophthalmology 2008;246:1575-1583. [20] Valtink M, Gruschwitz R, Funk RHW, Engelmann K. Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics. Cells Tissues Organs 2008;187:286-294. 24

[21] Akiyama

Y,

Kikuchi

isopropylacrylamide)

A,

Yamato

grafted

layer

M, on

Okano

T.

polystyrene

Ultrathin surfaces

poly(Nfor

cell

adhesion/detachment control. Langmuir 2004;20:5506-5511. [22] Zhang XZ, Wu DQ, Chu CC. Effect of the crosslinking level on the properties of temperature-sensitive

poly(N-isopropylacrylamide)

hydrogels.

Journal

of

Polymer Science Part B-Polymer Physics 2003;41:582-593. [23] Takahashi H, Matsuzaka N, Nakayama M, Kikuchi A, Yamato M, Okano T. Terminally functionalized thermoresponsive polymer brushes for simultaneously promoting

cell

adhesion

and

cell

sheet

harvest.

Biomacromolecules

2011;13:253-260. [24] Kwon OH, Kikuchi A, Yamato M, Okano T. Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes. Biomaterials 2003;24:1223-1232. [25] Ebara M, Yamato M, Aoyagi T, Kikuchi A, Sakai K, Okano T. Immobilization of cell-adhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest. Tissue Engineering 2004;10:1125-1135. [26] Schäfer-Soenen H, Moerkerke R, Berghmans H, Koningsveld R, Dusek K, Solc K. Zero and off-zero critical concentrations in systems containing polydisperse polymers with very high molar masses. 2. The system water-poly(vinyl methyl ether). Macromolecules 1997;30:410-416. [27] Gramm S, Teichmann J, Nitschke M, Gohs U, Eichhorn K-J, Werner C. Electron beam immobilization of functionalized poly(vinyl methyl ether) thin films on polymer surfaces – Towards stimuli responsive coatings for biomedical purposes. Express Polymer Letters 2011;5:970-976. [28] Hutter JL, Bechhoefer J. Calibration of atomic force microscope tips. Rev Sci Instrum 1993;64:1868-1873. [29] Nitschke M, Ricciardi S, Gramm S, Zschoche S, Herklotz M, Rivolo P et al. Surface

modification

of

cell

culture 25

carriers:

Routes

to

anhydride

functionalization of polystyrene. Colloids and Surfaces B: Biointerfaces 2012;90:41-47. [30] Gramm S, Komber H, Schmaljohann D. Copolymerization kinetics of Nisopropylacrylamide and diethylene glycol monomethylether monomethacrylate determined by online NMR spectroscopy. Journal of Polymer Science Part APolymer Chemistry 2005;43:142-148. [31] Nitschke M, Gramm S, Götze T, Valtink M, Drichel J, Voit B et al. Thermoresponsive poly(NiPAAm-co-DEGMA) substrates for gentle harvest of human corneal endothelial cell sheets. Journal of Biomedical Materials Research 2007;80A:1003-1010. [32] Nitschke M, Zschoche S, Baier A, Simon F, Werner C. Low pressure plasma immobilization of thin hydrogel films on polymer surfaces. Surface and Coatings Technology 2004;185:120-125. [33] Bednarz J, Teifel M, Friedl P, Engelmann K. Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells. Acta Ophthalmologica Scandinavica 2000;78:130-136. [34] Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y. Thermoresponsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromolecular Chemistry Rapid Communications 1990;11:571576. [35] Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science 2005;310:1139-1143. [36] Nicolas A, Geiger B, Safran SA. Cell mechanosensitivity controls the anisotropy of focal adhesions. Proceedings of the National Academy of Sciences of the United States of America 2004;101:12520-12525. [37] Sastry SK, Burridge K. Focal adhesions: A nexus for intracellular signaling and cytoskeletal dynamics. Exp Cell Res 2000;261:25-36.

26

[38] Gruschwitz R, Friedrichs J, Valtink M, Franz CM, Müller DJ, Funk RHW et al. Alignment and Cell-Matrix Interactions of Human Corneal Endothelial Cells on Nanostructured Collagen Type I Matrices. Investigative Ophthalmology & Visual Science 2010;51:6303-6310. [39] DiMilla PA, Stone JA, Quinn JA, Albelda SM, Lauffenburger DA. Maximal migration of human smooth muscle cells on fibronectin and type-IV collagen occurs at an intermediate attachment strength. Journal of Cell Biology 1993;122:729-737. [40] Le Saux G, Magenau A, Gunaratnam K, Kilian KA, Bocking T, Gooding JJ et al. Spacing of Integrin Ligands Influences Signal Transduction in Endothelial Cells. Biophysical Journal 2011;101:764-773. [41] Blake DA, Yu HN, Young DL, Caldwell DR. Matrix stimulates the proliferation of human corneal endothelial cells in culture. Investigative Ophthalmology & Visual Science 1997;38:1119-1129. [42] Pistsov MY, Sadovnikova EY, Danilov SM. Human corneal endothelial cells isolation, characterization and long-term cultivation. Experimental Eye Research 1988;47:403-414. [43] Yamaguchi M, Ebihara N, Shima N, Kimoto M, Funaki T, Yokoo S et al. Adhesion, migration, and proliferation of cultured human corneal endothelial cells by laminin-5. Investigative Ophthalmology & Visual Science 2011;52:679684. [44] Massia SP, Rao SS, Hubbell JA. Covalently immobilized laminin peptide TYRILE-GLY-SER-ARG (YIGSR) supports cell spreading and colocalization of the 67-kilodalton laminin receptor with alpha actinin and vinculin. Journal of Biological Chemistry 1993;268:8053-8059.

27

Figure Captions Figure 1: Preparation and biochemical functionalization of a thermo-responsive cell culture carrier (bottom) and a cell attaching to it (top): A glass carrier (1) is coated with polystyrene (2). A thin film of PVME-blend-PVMEMA is immobilized on the polystyrene surface by simultaneous electron beam cross-linking and grafting (3). Anhydride groups are formed in a thermal annealing step, which allows for covalent attachment of proteins or peptides containing free amino groups like cRGD (4). Subsequently, cells can attach to the surface by binding to the peptide with integrin receptors which leads to the formation of focal adhesions (5). Figure 2: HCEC after four days of cultivation at 37 °C on different PVME-blendPVMEMA samples (scale bar: 210 µm). Figure 3: HCEC after four days of cultivation at 37 °C on different PVME-blendPVMEMA samples and temperature reduction to 4 °C (scale bar: 210 µm). Figure 4: Metabolic activity rates of adherent HCEC determined with the WST-1 assay after four days of cultivation on PVME-blend-PVMEMA samples with a low and a high content of binding sites for the covalent immobilization of LN/ CS or cRGD, a thin or a thick layer, and a low or a high degree of cross-linking. Poly(NiPAAm-coDEGMA) and TCP with adsorbed LN/ CS or cRGD served as controls. Each data point represents three values gained during three independent measurements. Bar indicates standard deviation from mean. Figure 5: Immunocytochemical staining of adherent HCEC after four days of cultivation on PVME-blend-PVMEMA samples with a thin layer, a high degree of cross-linking and a high content of binding sites for the covalent immobilization of LN/ 28

CS or cRGD. Poly(NiPAAm-co-DEGMA) and TCP with adsorbed LN/CS or cRGD served as controls. Paxillin associated to focal adhesions (A to F, white arrowheads), fibronectin as part of the deposited ECM (G to L), and ZO-1 associated to tight junctions (M to R) are shown in green, F-actin fibers in red (Phalloidin) and the nuclei in blue (Hoechst). Figure 6: Concept for the design of tunable thermo-responsive cell culture carriers.

29

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6